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Paracoccus denitrificans: a genetically tractable model system for studying respiratory complex I.

ABSTRACT: Mitochondrial complex I (NADH:ubiquinone oxidoreductase) is a crucial metabolic enzyme that couples the free energy released from NADH oxidation and ubiquinone reduction to the translocation of four protons across the inner mitochondrial membrane, creating the proton motive force for ATP synthesis. The mechanism by which the energy is captured, and the mechanism and pathways of proton pumping, remain elusive despite recent advances in structural knowledge. Progress has been limited by a lack of model systems able to combine functional and structural analyses with targeted mutagenic interrogation throughout the entire complex. Here, we develop and present the α-proteobacterium Paracoccus denitrificans as a suitable bacterial model system for mitochondrial complex I. First, we develop a robust purification protocol to isolate highly active complex I by introducing a His₆-tag on the Nqo5 subunit. Then, we optimize the reconstitution of the enzyme into liposomes, demonstrating its proton pumping activity. Finally, we develop a strain of P. denitrificans that is amenable to complex I mutagenesis and create a catalytically inactive variant of the enzyme. Our model provides new opportunities to disentangle the mechanism of complex I by combining mutagenesis in every subunit with established interrogative biophysical measurements on both the soluble and membrane bound enzymes.

SUBMITTER: Jarman OD

PROVIDER: S-EPMC8115037 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Two-component system that regulates methanol and formaldehyde oxidation in Paracoccus denitrificans.

Project description:A chromosomal region encoding a two-component regulatory system, FlhRS, has been isolated from Paracoccus denitrificans. FlhRS-deficient mutants were unable to grow on methanol, methylamine, or choline as the carbon and energy source. Expression of the gene encoding glutathione-dependent formaldehyde dehydrogenase (fhlA) was undetectable in the mutant, and expression of the S-formylglutathione hydrolase gene (fghA) was reduced in the mutant background. In addition, methanol dehydrogenase was immunologically undetectable in cell extracts of FhlRS mutants. These results indicate that the FlhRS sensor-regulator pair is involved in the regulation of formaldehyde, methanol, and methylamine oxidation. The effect that the FlhRS proteins exert on the regulation of C(1) metabolism might be essential to maintain the internal concentration of formaldehyde below toxic levels.

| S-EPMC94923 | biostudies-literature

Exploring the Denitrification Proteome of Paracoccus denitrificans PD1222.

Project description:Denitrification is a respiratory process that produces nitrous oxide as an intermediate, which may escape to the atmosphere before its reduction to dinitrogen through the nitrous oxide reductase NosZ. In this work, the denitrification process carried out by Paracoccus denitrificans PD1222 has been explored through a quantitative proteomic analysis. Under anaerobic conditions, with nitrate as sole nitrogen source, the synthesis of all the enzymes involved in denitrification, the respiratory nitrate, nitrite, nitric oxide, and nitrous oxide reductases, was increased. However, the periplasmic and assimilatory nitrate reductases decreased. Synthesis of transporters for alcohols, D-methionine, sulfate and copper, most of the enzymes involved in the tricarboxylic acid cycle, and proteins involved in other metabolic processes like lysine catabolism, fatty acids degradation and acetyl-CoA synthesis, was increased during denitrification in P. denitrificans PD1222. As consequence, an enhanced production of the central metabolite acetyl-CoA was observed. After establishing the key features of the denitrification proteome, its changes by the influence of a competitive electron acceptor, oxygen, or competitive nitrogen source, ammonium, were evaluated.

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Analyzing fission yeast multidrug resistance mechanisms to develop a genetically tractable model system for chemical biology.

Project description:Chemical inhibitors can help analyze dynamic cellular processes, particularly when probes are active in genetically tractable model systems. Although fission yeast has served as an important model system, which shares more cellular processes (e.g., RNAi) with humans than budding yeast, its use for chemical biology has been limited by its multidrug resistance (MDR) response. Using genomics and genetics approaches, we identified the key transcription factors and drug-efflux transporters responsible for fission yeast MDR and designed strains sensitive to a wide-range of chemical inhibitors, including commonly used probes. We used this strain, along with acute chemical inhibition and high-resolution imaging, to examine metaphase spindle organization in a "closed" mitosis. Together, our findings suggest that our fission yeast strains will allow the use of several inhibitors as probes, discovery of new inhibitors, and analysis of drug action.

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Transcriptome analysis of Paracoccus denitrificans during Cu-limitation

2013-11-18 | GSE48577 | GEO

Transcriptome analysis of Paracoccus denitrificans during Cu-limitation

Project description:Transcriptional profiling of Paracoccus denitrificans PD1222 wild type grown to mid-exponential phase in minimal media with either 13 uM (Cu-H) or 0.5 uM (Cu-L) Cu regimes. The goal was to define the effects of Cu-limitation on denitrification genes Two growth conditions, three biological replicates of each condition. Each sample hybridised in a two-channel hybridization against Paracoccus denitrificans genomic DNA as the comparator/reference, which also acted as a control for spot quality. Cu-concentration 13 uM (Cu-H) versus 0.5 uM Cu (Cu-L) in anaerobic growth conditions.

2013-11-18 | E-GEOD-48577 | biostudies-arrayexpress

Characterization of a TIR-like protein from Paracoccus denitrificans.

Project description:Based on protein sequence homology searches, we found a conserved open reading frame within the genome of several human pathogenic bacteria showing a resemblance to the mammalian TIR domain. We cloned, expressed, and characterized the corresponding gene product from Paracoccus denitrificans using several biophysical techniques. The protein consists of two independently folded domains. As predicted from the amino acid sequence and experimentally confirmed here, the N-terminal domain consists of a alpha-helical coiled-coil. The NMR data indicates that the C-terminal TIR-like domain folds into a compact protein. Finally, using GST pull-down experiments, we show that the bacteria TIR-like domain binds to the mammalian receptor (TLR4) and adaptor (MyD88) TIR domains. We postulate that prokaryotic pathogens utilize the TIR-like proteins to interfere with the innate immune response of the mammalian host so that the bacterial infection can progress undetected.

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Zinc-Dependent Transcriptional Regulation in <i>Paracoccus denitrificans</i>.

Project description:Zinc homeostasis is critical for bacterial survival and is mediated largely at the transcriptional level by the regulation of zinc uptake and efflux genes. Here we use RNA-seq to assess transcriptional changes as a result of zinc limitation in the denitrifying bacterium Paracoccus denitrificans. The results identify the differential expression of 147 genes, most of which were upregulated in zinc-depleted medium. Included in this set of genes are a large number of transition metal transporters, several transcription factors, and hypothetical proteins. Intriguingly, genes encoding nitric oxide reductase (norCB) and nitrite reductase (nirS) were also upregulated. A Zur consensus binding motif was identified in the promoters of the most highly upregulated genes. The zinc uptake regulator (Zur) from this organism was also characterized and shown to bind to the Zur motif in a zinc-dependent manner. This work expands our current understanding of the transcriptional response of gram-negative bacteria to zinc limitation and identifies genes involved in denitrification as part of the Zur regulon.

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Transcriptome sequencing of Paracoccus denitrificans ATCC19367 with florfenicol exporsure

Project description:We report the application of transcriptome sequencing technology in florfenicol interfering with denitrification by Paracoccus denitrificans. More than 30 billion bases of sequences were obtained by sequencing analysis. We found 433 differentially expressed genes, of which 292 genes were down-regulated and 141 genes were up-regulated, respectively. Importantly, most of the denitrification genes were suppressed, which further led to the enrichment of key metabolic pathways. The weakening of protein synthesis was consistent with the bacteriostatic mechanism of florfenicol. In particular, we found 42 putative differentially expressed sRNAs. After homologous alignment, target gene prediction and functional analysis, it was proved that the sRNAs differential expression profile is likely to be a key transcription factor affected by antibiotics in denitrification. This study provides new ideas for further control of environmental antibiotic pollution.

2021-04-13 | GSE166910 | GEO

Proteome-wide dataset generated by iTRAQ-3DLCMS/MS technique for studying the role of FerB protein in oxidative stress in Paracoccus denitrificans.

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